Molecular mechanisms of NMDA excitotoxicity in the retina

NMDA excitotoxicity, as a part of glutamate excitotoxicity, has been proposed to contribute significantly to many retinal diseases. Therefore, understanding mechanisms of NMDA excitotoxicity will provide further insight into the mechanisms of many retinal diseases. To study mechanisms of NMDA excitotoxicity in vivo, we used an animal model in which NMDA (20 mM, 2 µL) was injected into the vitreous of mice. We also used high-throughput expression profiling, various animals with reduced expression of target genes, and animals treated with the oral iron chelator deferiprone. We found that the expression of many genes involved in inflammation, programmed cell death, free radical production, oxidative stress, and iron and calcium signaling was significantly increased 24 h after NMDA treatment. Meanwhile, decreased activity of the pro-inflammatory TNF signaling cascade and decreased levels of ferrous iron (Fe2+, required for free radical production) led to significant neuroprotection in NMDA-treated retinas. Since increased TNF signaling activity and high Fe2+ levels trigger regulated necrosis, which, in turn, lead to inflammation, we proposed an important role in NMDA excitotoxicity of a positive feedback loop in which regulated necrosis promotes inflammation, which subsequently triggers regulated necrosis.

The retina contains a variety of excitatory retinal neurons whose main neurotransmitter is glutamate 1 .Thus, it is not surprising that retinal damage due to disease (e.g., glaucoma and ischemic optic neuropathy) or injury results in the release of significant amounts of glutamate into the extracellular space [2][3][4][5] .Extracellular glutamate is not a passive witness to the developing pathology in the retina, as it plays a significant and active role.Glutamate neurotransmission is tightly regulated by an array of receptors and transporters to minimize the presence of excess glutamate in the extracellular space 6 .Meanwhile, high levels of extracellular glutamate lead to excitotoxicity, an important factor of many neurodegenerative diseases 6 .Within the retina, glutamate excitotoxicity can lead to a significant deterioration in vision and, in some cases, even blindness [2][3][4][5][6] .One of the mechanisms leading to glutamate excitotoxicity is NMDA receptor overactivation, which is known as NMDA excitotoxicity 6 .The objective of this study was to investigate mechanisms of NMDA excitotoxicity in the retina.
Retinal ganglion cells (RGCs) are the only retinal neurons that send their axons to the visual cortex of the brain 1 .RGC death due to disease or injury ultimately leads to blindness [2][3][4][5][6] .At the same time, amacrine cells modulate the signal transmitted from bipolar cells to RGCs within the inner plexiform layer and modulate the activity of RGCs within the ganglion cell layer 7 .High NMDA levels in the retina lead to the death of RGCs and amacrine cells [8][9][10][11] .However, the dynamics of the death of these neurons is different: amacrine cells begin to die first, while the death of RGCs is delayed [8][9][10][11] .It was also observed that murine amacrine cells die predominantly via necrosis during the first hour, while apoptosis of these cells is observed only by the third hour after NMDA treatment 8,9 .The death of RGCs in the presence of NMDA is a more complex problem.While the significant RGC death in the presence of NMDA is well documented in vivo, there is uncertainty as to whether NMDA leads to significant RGC death in vitro [8][9][10][11][12] .The results obtained in Dr. Barres' laboratory suggest that NMDA is either non-toxic or even promotes the survival of RGCs in vitro 9,12 .Other studies suggest that NMDA causes RGC death in vitro 13 .However, even so, these neurons are probably less sensitive to NMDA excitotoxicity in vitro than other types of neurons 9,12,13 .Thus, it is not entirely clear whether RGC death in vivo is a result of overactivation of NMDA receptors or an indirect effect of NMDA excitotoxicity.The findings from this study suggest that NMDA-induced inflammation results in the activation of TNF signaling and an increase in ferrous (Fe 2+ ) iron levels, leading to RGC death.Thus, the mechanism of NMDA excitotoxicity is more complex and is determined by the direct and indirect effects of NMDA on retinal neurons.

Results
The activity of retinal NMDA receptors leads to significant changes in gene expression accompanied by RGC death RGCs and displaced amacrine cells are found only in the ganglion cell layer (GCL) of the retina in a relatively equal proportion 14 .In their paper published in 2004, Ullian et al. showed that RGCs are not sensitive to glutamate and NMDA excitotoxicity, while amacrine cells are very sensitive and die quickly in the presence of glutamate and NMDA 9 .Before and after this publication, there was much evidence that high levels of extracellular glutamate and NMDA are toxic to RGCs, leading to their death [2][3][4][5][6]8,10,11,13 . However, te presence of this publication has led to more careful consideration of which cell types in the retina are more sensitive to glutamate and NMDA excitotoxicity.To study the effects of extracellular NMDA on the survival of neurons in the GCL of the retina, we injected NMDA (20 mM, 2 µL; n = 6) and phosphate buffered saline (PBS as a control, 2 µL; n = 6) into the vitreous of the wild type (WT) mice.NMDA was injected into the left eyes of the animals and PBS was injected into the right eyes of the same animals.To determine the number of surviving neurons and RGCs in the GCL, the retinas of these animals were collected 7 days after treatment, and whole retina flat mounts were stained using a neuronal marker Tubb3 and a RGC marker Rbpms (Fig. 1A).The percentage of surviving cells was determined for each animal as the ratio of the mean number of cells counted in the NMDA-treated left eye to the mean number of cells counted in the PBS-treated right eye.The mean values obtained for a group of six animals for each of the studied cell markers are shown in Fig. 1A.We found that only 25 ± 2% (n = 6) of the Tubb3-positive GCL neurons and 19 ± 2% (n = 6) of the Rbpms-positive RGCs survived the NMDA treatment (Fig. 1A).Since Tubb3-positive GCL neurons correspond to the entire population of surviving neurons (RGCs and displaced amacrine cells), the percentage of surviving amacrine cells should be about 6%.This number of surviving amacrine cells is significantly less than the number of surviving RGCs, indicating that amacrine cells are more sensitive to NMDA excitotoxicity than RGCs.
To study the effects of NMDA excitotoxicity at the molecular level, we injected NMDA (20 mM, 2 µL; n = 4) and PBS (2 µL; n = 4) into the vitreous of the WT mice.The retinas of these animals were collected 24 h after treatment and used in RNA-seq analysis to examine changes in gene expression (Supplementary Fig. S1).To reach a significant depth of sequencing and thus obtain information on all types of retinal cells and not just on photoreceptors, which make up 70% of all retinal cells, we sequenced 52,309,779 ± 4,094,028 fragments (or more than 100 M reads) on average per library among which 40,441,184 ± 2,887,342 fragments were uniquely mapped to the mouse genome.The differential expression analysis of our RNA-seq data indicates that gene expression in NMDA-treated retinas is significantly altered compared to PBS-treated control retinas.This follows from the value of the correlation coefficient, MA and volcano plots, sample clustering, and principal component analysis (PCA) (Fig. 1B-F).The number of genes whose expression was statistically significantly (p-value adjusted [padj] < 0.05) increased by two or more times (log2 fold change [log2FC] ≥ 1) was 1422, while the number of genes whose expression was statistically significantly (padj < 0.05) reduced by two or more times (log2FC ≤ − 1) was 1352 (Fig. 1G, Supplementary Data S1).This data indicates that NMDA excitotoxicity has a significant impact on gene expression in the retina 24 h after treatment.

NMDA excitotoxicity is accompanied by an increased expression of genes of signaling cascades, the high activity of which is dangerous for retinal neurons
Significant changes in the expression of many genes do not allow us to understand the impact of these changes until they are attributed to specific processes and signaling cascades.To this end, we used Gene Set Enrichment Analysis (GSEA), a powerful analytical method for interpreting RNA-seq data 15 .The results of the GSEA analysis indicated that most biological processes and signaling cascades whose gene expression is significantly upregulated 24 h after NMDA treatment fall into the following groups: (1) inflammation, (2) programmed cell death; (3) reactive oxygen and nitrogen species (ROS/RNS) production and oxidative stress, (4) iron signaling, and (5) calcium signaling (Fig. 2A, Supplementary Data S2).The biological processes and signaling cascades presented in the first (1) group indicate the important role of the innate immune system in general and such cascades as TNF signaling, IL1b signaling, toll like receptor (TLR) signaling, and interferon signaling, in particular (Fig. 2A).The second (2) group includes not only apoptosis but also regulated necrosis (e.g., necroptosis and pyroptosis).The connection of this group with the third (3) group should also be noted (GOBP Positive Regulation of Oxidative Stress Induced Cell Death [FDR q-val = 0.015], GOBP Regulation of Oxidative Stress Induced Cell Death [FDR q-val = 0.083], Fig. 2A).The third (3) group reflects an increased expression of genes, the activity of which may lead to a significant production of ROS/RNS with subsequent oxidative stress.We note the dependence of the third (3) group on the fourth (4) group, since the increased activity of biological processes and signaling cascades belonging to the fourth (4) group may lead to ferrous (Fe 2+ ) iron accumulation.Ferrous (Fe 2+ ) iron is a catalyst for the Fenton/Haber-Weiss reaction leading to the production of large amounts of free radicals (ROS/RNS).This suggests that oxytosis/ferroptosis, a type of regulated necrosis dependent on high free radical and ferrous (Fe 2+ ) iron levels, may also be involved in NMDA excitotoxicity.
The results of our GSEA analysis suggest that free radicals and ferrous (Fe 2+ ) iron as a catalyst for their production contribute to NMDA excitotoxicity.Evidence in favor of this hypothesis is the increased expression of genes such as Steap3 (log2FC = 2.28, padj = 3.9•10 -14 ), Steap4 (log2FC = 1.59, padj = 1.5•10 -4 ), Ftl1 (log2FC = 1.37, padj = 1.9•10 -10 ), and Slc25a37 (Mfrn1; log2FC = 2.09, padj = 6.6•10 -8 ) in NMDA treated retinas (Fig. 3E, Supplementary Data S1).Of note is the increased expression of Steap3: the enzyme it encodes is responsible for the generation of ferrous (Fe 2+ ) iron from ferric (Fe 3+ ) iron 25 .To evaluate the significance of these data, we used the oral iron chelator deferiprone (DFP).It has previously been shown that DFP significantly reduces the level of www.nature.com/scientificreports/labile iron in the retina, protecting it from many pathologies [26][27][28][29][30][31] .We expected that lowering labile iron would lead to reduced NMDA excitotoxicity.To this end, we used two groups of animals: one was treated with DFP (1 mg/mL in drinking water; animals were given fresh DFP daily) and the other was not treated and served as a control (untr).Because a mouse drinks an average of 5 mL of water per day, our experimental mice consumed an average of 5 mg of DFP daily.Animals were pre-treated with DFP for 8 days prior to NMDA injection and were treated with DFP for 7 days after injection (Supplementary Fig. S1).After this period, the retinas of the experimental and control animals were collected and used to count the surviving GCL neurons and RGCs.We found that the quantity of RGCs in DFP-treated animals was significantly higher compared to untreated controls (Tubb3: 44 ± 4%

Discussion
Overactivation of NMDA receptors makes a significant contribution to glutamate excitotoxicity and is known as NMDA excitotoxicity [8][9][10][11]13 . To tudy mechanisms of NMDA excitotoxicity in vivo, we used an animal model in which NMDA was injected into the vitreous of mice.We also used RNA-seq analysis, knockout animals, and animals treated with an iron chelator.The results of our RNA-seq analysis indicate activation of many signaling cascades involved in inflammation, programmed cell death, free radical production, oxidative stress, and iron and calcium metabolism 24 h after NMDA treatment.Meanwhile, the expression of genes whose activity is necessary to maintain normal neuronal function was reduced.Our data indicate an important role for the TNF signaling cascade and ferrous (Fe 2+ ) iron production in retinal NMDA excitotoxicity.We found some neuroprotection upon inactivation of Gsdmd, whose activity leads to inflammasome-dependent inflammation and regulated necrosis.However, this neuroprotection was less pronounced compared to the neuroprotection that occurs when TNF signaling was inactivated or when ferrous (Fe 2+ ) iron level was reduced.NMDA excitotoxicity leads to death of RGCs and displaced amacrine cells in the ganglion cell layer and amacrine cells in the inner nuclear layer 8,9 .Significant necrosis of amacrine cells is observed already within the first hour after NMDA treatment 9 .Amacrine cell apoptosis can be detected only by the third hour after NMDA treatment 8,9 .At the same time, published data and our results presented here indicate that RGCs are more resistant than amacrine cells to NMDA excitotoxicity.The in vitro results obtained in Dr. Barres' laboratory indicate that the presence of glutamate or NMDA in the cell culture medium either does not lead to the RGC death, or it even promotes their survival 9,12 .At the same time, the results of many investigators, including our data, indicate a significant loss of RGCs after NMDA treatment in vivo [2][3][4][5][6]8,10,11,13 .However, the RGCs began to die much later than amacrine cells, that is, their death was delayed [8][9][10][11] .How could this contradiction be explained?It is an established fact that cell death via necrosis leads to a significant inflammatory response in the tissue [16][17][18][19][20] .Many damage-associated molecular patterns (DAMPs: e.g., Hsp70 and Hmgb1) released from necrotic cells activate the same pattern recognition receptors (Tlr4 as an example) as products of pathogens, resulting in a strong inflammatory response [16][17][18][19][20][32][33][34] .The secretion of the Tnf cytokine is one of the critical events in the inflammatory response 35 .Our RNA-seq data indicate a significant inflammatory response in the retina 24 h after NMDA treatment.Necrosis of amacrine cells in the first hours after NMDA treatment could explain the occurrence of such a strong inflammatory response.Our data also provide evidence of activation of the TNF signaling cascade 24 h after NMDA treatment.Meanwhile, inactivation of this cascade leads to significant RGC survival in NMDA treated retinas.Our data and the results of other investigators indicate that there is significant RGC death in the presence of the Tnf cytokine in vivo and in vitro 8,[22][23][24]31,[36][37][38][39] .It has also been shown that inhibition of this cytokine leads to significant RGC survival after NMDA treatment 8 .The totality of these data allows us to propose the following mechanism.Necrosis of amacrine cells in the first hours after NMDA treatment leads to an inflammatory response, including Tnf production and secretion.In turn, high Tnf levels lead to the death of RGCs, which could explain their delayed death.It should be noted that Tnf leads to cell death not only via apoptosis, but also via regulated necrosis known as necroptosis 35 .Our RNA-seq data indicate an increased expression of Ripk1, Ripk3, and Mlkl genes that trigger necroptosis 35 .Thus, RGC and amacrine cell regulated necrosis could lead to increased inflammatory reaction by launching a positive feedback loop in which regulated necrosis promotes inflammation and inflammation triggers regulated necrosis.All this together can result in significant RGC death in NMDA treated retinas.
The results of our study indicate that free radicals (ROS/RNS) and ferrous (Fe 2+ ) iron as a catalyst for their production contribute to NMDA excitotoxicity.By lowering the level of labile iron in the NMDA-treated retinas using the oral iron chelator DFP, we were able to achieve significant RGC survival.Increased levels of free radicals in the NMDA-treated retinas and their negative impact on RGC survival have been shown previously [3][4][5][6]40,41 . Howevr, the contribution of ferrous (Fe 2+ ) iron to retinal NMDA excitotoxicity has been examined in only one study by Sakamoto et al. to the best of our knowledge 42 .In agreement with our data, these authors showed increased labile ferrous (Fe 2+ ) iron, free radical, and oxidative stress levels in NMDA-treated retinas 42 .At the same time, the authors showed a decrease in labile ferrous (Fe 2+ ) iron and oxidative stress levels and an increase in the level of surviving RGCs in the NMDA-and iron chelator-treated retinas 42 .These findings suggest an important role for ferrous (Fe 2+ ) iron as a catalyst for free radical production in retinal NMDA excitotoxicity.These data also show that since oxytosis/ferroptosis depends on high ferrous (Fe 2+ ) iron and free radical levels, this type of regulated necrosis should be involved in NMDA excitotoxicity 43,44 .Thus, oxytosis/ferroptosis, together with necroptosis, would contribute to maintaining the positive feedback loop described in the previous paragraph leading to significant RGC death in the NMDA-treated retinas.www.nature.com/scientificreports/Inflammasome assembly is required to activate caspase-1, which cleaves pro-Il1b to generate the mature cytokine and cleaves gasdermins (GSDM) to generate pore-forming fragments that, in turn, targets the membrane and allows the release of mature Il1b 18,45 .However, the presence of a large number of GSDM pores on the cell membrane over a long period of time can lead to cell death via regulated necrosis, known as pyroptosis 18,45 .Thus, inflammasome assembly can lead to both inflammation and pyroptosis in the tissue. Th results of our GSEA analysis suggested a contribution of inflammasome activity and pyroptosis to retinal NMDA excitotoxicity.Since GSDM pore formation is one of the key events leading to the inflammatory response and pyroptosis, we used Gsdmd deficient (GsdmdKO) animals to evaluate the role of these processes in NMDA excitotoxicity.While the percentage of surviving RGCs was higher in GsdmdKO mice compared to WT controls, this value was significantly lower than the values obtained in TNFR1KO animals or in iron chelator-treated animals (Fig. 3).These data suggest that the inflammasome role is probably less significant than the role of TNF signaling and ferrous (Fe 2+ ) iron in retinal NMDA excitotoxicity.Tsoka et al. came to a similar conclusion when they examined the contribution of inflammasome-mediated inflammation to retinal NMDA excitotoxicity 46 .
In conclusion, the results of our study and previously published data support a mechanism of retinal NMDA excitotoxicity in which overactivation of NMDA receptors leads to rapid death of amacrine cells via necrosis (Fig. 4).In turn, DAMPs released from necrotic amacrine cells trigger a strong inflammatory response, including the activation of TNF signaling.The Tnf cytokine is toxic to RGCs and, thus, high Tnf levels lead to RGC death through apoptosis and necroptosis (one of the types of regulated necrosis).This mechanism can explain the delayed death of RGCs in retinal NMDA excitotoxicity.We do not rule out that Tnf may also lead to the death of remaining amacrine cells through apoptosis and necroptosis.Our results and previously published data also suggest that ferrous (Fe 2+ ) iron-dependent regulated necrosis (oxytosis/ferroptosis) of RGCs and amacrine cells contributes to NMDA excitotoxicity.In turn, RGC and amacrine cell necroptosis and oxytosis/ferroptosis should promote inflammation and retinal damage by launching the positive feedback loop in which regulated necrosis promotes inflammation, which subsequently triggers regulated necrosis (Fig. 4).The proposed mechanism provides insight into retinal NMDA excitotoxicity.Since NMDA excitotoxicity is part of glutamate excitotoxicity, which, in turn, is an important contributor to many retinal diseases, our proposed mechanism highlights several targets for drug development that could help patients suffering from such retinal diseases.

Animals and ethics statement
All procedures were executed in compliance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and according to the University of Miami Institutional Animal Care and Use Committee (IACUC) approved protocol (Protocol #: 21-070).TNFR1 and Gsdmd knockout animals (TNFR1KO and GsdmdKO, respectively; these knockouts have the C57BL/6 J genetic background) and C57BL/6J mice as the wild-type (WT) controls were received from the Jackson Laboratory (Bar Harbor, ME, USA; stock numbers 003242, 032663, and 000664).The oral iron chelator deferiprone (DFP, 1 mg/mL, #379409, MilliporeSigma, St. Louis, MO, USA) was delivered to animals via drinking water.We used 2-4-month-old male and female mice to address sex as a biological variable.Animals were housed under standard conditions of humidity and temperature, were given free access to food and water, and had a 12-h light to dark cycle.All methods were completed and reported in accordance with ARRIVE guidelines.

Animal model of NMDA excitotoxicity
The animals were anaesthetized by intraperitoneal injection of ketamine (80 mg/kg)/xylazine (10 mg/kg) to perform intravitreal injections.We used change in heart rate in response to tail pinch and corneal reflex as an indication of the level of anesthesia.Intravitreal injections were performed under a microsurgical microscope using glass pipettes with a diameter of approximately 150 µm at the tip.Each eye was punctured at the upper nasal limbus and a volume of 2 µL of NMDA (20 mM, M3262, MilliporeSigma, St. Louis, MO, USA) or

Figure 1 .
Figure 1.Retinal NMDA excitotoxicity is accompanied by significant changes in gene expression.(A) NMDA injection into the vitreous leads to significant neuronal death in the ganglion cell layer (GCL) after 7 days.(B) Correlation (Corr) value and FPKM distributions were generated to visualize the correlation between levels of gene expression in NMDA-treated and control (PBS-treated) retinas 24 h after treatment.(C,D) MA and volcano plots show dramatic and statistically significant changes in gene expression after NMDA treatment.padj is p value adjusted; lfc is the log2 fold change (Log2FC) between two conditions (NMDA vs. PBS).(E,F) A heatmap of the sample-to-sample distances (sample clustering, E) and the principal component analysis (PCA) plot of the samples (F) illustrate the significant difference between NMDA-treated and PBS-treated retinas.(G)The table provides examples of genes whose expression is increased (highlighted in dark red) and whose expression is decreased (highlighted in green) in the NMDA-treated retinas.Log2FC is the logarithm (log2) of the fold change in gene expression (NMDA vs. PBS).This is a standard approach for characterizing gene expression in bioinformatics.To get the usual value of changes in gene expression, this formula (2 Log2FC ) should be used.

Figure 2 .
Figure 2. Gene expression analysis revealed a diversity of signaling cascades and biological processes triggered by NMDA in the retina.(A) NMDA increases the expression of genes involved in signaling cascades and biological processes whose activity can lead to retinal damage.At the same time, NMDA reduces the expression of genes whose activity is necessary for normal retinal function.(B) The table shows examples of genes involved in calcium signaling whose expression is not only increased but also decreased in NMDA-treated retinas.

Figure 3 .
Figure 3. Signaling cascades controlling inflammation and regulated necrosis contribute significantly to NMDA excitotoxicity.(A) While the Tnf cytokine triggers an inflammatory response by activating TNFR1 receptors on the surface of glial cells (astrocytes and microglia), it can trigger RGC regulated necrosis (necroptosis) by activating TNFR1 receptors on their surface, followed by phosphorylation of Ripk1, Ripk3, and Mlkl.In turn, damage associated molecular patterns (DAMPs) released from necrotic cells activate pattern recognition receptors such as Tlr4 on the surface of glial cells, enhancing the inflammatory response.The expression of the genes highlighted in dark red is increased.(B) TNFR1 inactivation reduces NMDA excitotoxicity (**p-value < 0.01).(C) The expression of many genes involved in the inflammasome pathway is significantly increased in NMDA-treated retinas.The activity of this pathway leads to inflammation and regulated necrosis (pyroptosis).(D) Inactivation of one of the key genes, Gsdmd, in the inflammasome pathway increases the survival of RGCs in the NMDA-treated retinas (*p-value < 0.05).(E) Increased expression of iron signaling genes suggests ferrous (Fe 2+ ) iron accumulation in NMDA-treated retinas.(F) Lowering iron levels using the oral iron chelator deferiprone (DFP) significantly increases RGC survival in the NMDA-treated retinas.

Figure 4 .
Figure 4.This figure illustrates our proposed mechanism for retinal NMDA excitotoxicity.According to this mechanism, NMDA-mediated amacrine cell (AC) death via necrosis triggers a positive feedback loop, leading to significant RGC death and retinal degeneration.